Molecular design of dual-emission rhodamine analogs

Abstract

Rhodamine derivatives are one of the most important classes of fluorophores, exhibiting a unique equilibrium between a fluorescent open form and a (nearly) non-emissive closed form. The closed form has been understudied, due to its short wavelength and low quantum yield. In this work, through detailed quantum chemical calculations, we showed that closed-form rhodamines are non-fluorescent, because a weakly emissive charge transfer (CT) state is more stable than the virtually emissive locally excited (LE) state. We also proposed a rapid design method to activate the fluorescence of rhodamine analogs in the closed form, via a judicious choice of three molecular fragments with matched frontier molecular orbitals to stabilize the LE state. We foresee that the resulting dual emissions (from both the closed and open forms of these analogs) can be used for constructing ratiometric probes and dual-channel fluorescent labels for advanced bioimaging and biosensing applications.

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Introduction

Rhodamine and derivatives (henceforth denoted as rhodamines) represent one of the most important classes of organic fluorophores.^1,2 Ever since their discovery, these dyes have been widely used in numerous bioimaging^3–5 and biosensing applications.^6–8 This is largely due to their high absorption coefficients, good fluorescence quantum yields, excellent photostability, and superior biocompatibility.^9–11 Another interesting aspect of rhodamine dyes is that they exist in an equilibrium between an almost non-emissive, spirocyclic form and a fluorescent zwitterionic/cation form (Scheme 1a).^12–14 Extensive studies have been conducted to optimize or utilize the fluorescent properties in the open form of rhodamines. Nevertheless, less attention is directed to understanding the weakly emissive nature of the closed form, let alone methods of turning on bright fluorescence in the closed form.

Scheme 1 (a) Illustration of spirocyclization equilibrium between the closed form and the open form of rhodamines. (b) The applications of single-emission rhodamines and potential applications of dual-emission rhodamine analogs. (c) The molecular design strategy of turning closed-form rhodamine analogs emissive (L: LUMO; H: HOMO).

A plethora of important research work has been conducted to tune the fluorescent properties of the open form of rhodamine derivatives. Replacing the oxygen-bridging atom in the xanthene scaffold with other heteroatoms (such as C, Si, S, and P) leads to a platter of colourful rhodamines with emissions spanning the visible to NIR spectrum.^15–19 Changing the dialkylamino group with azetidine or several other amino groups could greatly enhance the quantum yields and the photostability of the open-form rhodamines by inhibiting twisted intramolecular charge transfer (TICT).^20–24 These colourful, bright, and photostable rhodamines have been deployed in many critical bioimaging experiments.^25–30 Additionally, modulating the equilibrium between the closed and the open forms of rhodamines has been exploited to develop fluorescent turn-on probes that detect various species.^31,32 Optimizing this equilibrium also endows several hydroxymethyl rhodamines with spontaneously blinking properties, enabling long-lasting super-resolution imaging based on the single-molecule localization technique.^33,34

Less emphasis has been placed on their closed-form counterparts, mainly due to their short UV-vis absorption and emission wavelengths and low fluorescence quantum yields. Notably, Klein and Hafner reported that Rhodamine B exhibits dual emissions, attributed to the emissions from both the closed and open forms.³⁵ The closed-form emissions have a strong polarity dependence and have been assigned to a charge-transfer (CT) state. The CT emissions appear in the short wavelength region (λ_em < 500 nm) with a low quantum yield (up to 5.2% in dioxane). Similarly, Karpiuk and co-workers discovered that the closed-form of Rhodamine 101 experiences considerable CT upon photoexcitation and exhibits a weak and broad CT emission band peaked at ∼465 nm in dibutyl ether (quantum yield φ = 2.3%).¹³ However, the low quantum yields and the short excitation/emission wavelengths (UV-vis absorption <350 nm) of the closed form are of low practical utility, especially for bioimaging and biosensing applications. Moreover, the weakly emissive nature of the closed form is often attributed to the disruption of the π-conjugation in the chromophore scaffold.^11,14,36 Yet, few studies provide a deep mechanistic understanding of the electronic structures or whether the close form can become fluorescent at favourable long wavelengths. Understanding these molecular origins, however, could light up both the closed and open forms of rhodamines, thus enabling many promising applications (i.e., the construction of ratiometric sensors and the development of dual-channel fluorophores for super-resolution imaging; Scheme 1b).

Herein, we reported the theoretical investigation that explains why the closed form of rhodamines is non-fluorescent (Scheme 1c). Based on these results, we also reported a facile screening method to activate intense closed-form fluorescence and design dual-emission rhodamine analogs.

Computation methods

All density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations were carried out using the Gaussian 16A suite of programs.³⁷ Geometry optimizations were carried out at the CAM-B3LYP/def2SVP level.³⁸ Solvation effects were taken into account using the SMD model,³⁹ using water as the solvent. The excitation and emission energies of all molecules were calculated using the corrected linear-response (cLR) solvent formalism. Frequency calculations were performed to confirm that we obtained stable structures without imaginary vibration frequencies unless stated otherwise. For the optimization of the CT states, we applied constraints to lock the dihedral angles in various rhodamines to maximize charge separation (please see the ESI† for computational details and justifications). The molecular excitation properties were also investigated by the hole–electron analysis using Multiwfn 3.6.⁴⁰

Results and discussion

Molecular origins of weak emissions in the closed form of conventional rhodamine derivatives

To understand why the closed form of conventional rhodamine dyes (Fig. 1a) is weakly emissive, we first performed detailed calculations on DRHN1 (Fig. 1b). Our computational results revealed that after geometrical relaxation in the excited state, the CT state of DRHN1 is the most stable state with a relative energy level of 3.280 eV and small oscillator strength of f = 0.027. This small f value is mainly caused by the poor overlap between the hole and the electron in the CT state. Such a small f value indicates that the CT state is (nearly) non-emissive.

Fig. 1 (a) Molecular structures of conventional rhodamines. (b) The energy levels in the adiabatic excited states in water (the top panel), and the corresponding electron and hole distributions (the bottom panel) of DRHN1. (c) The oscillator strength of the CT states and (d) the calculated ΔE_LE–CT values of rhodamines as shown in (a).

At the same time, we noted a locally excited (LE) state (4.245 eV) whose energy level is 0.965 eV higher than that of the CT state. The electron and hole of the LE state reside in the amino-phenyl moiety of DRHN1, affording a large f value of 0.245. The large f value suggests that this LE state is virtually highly emissive. However, DRHN1 will quickly relax to the CT state via internal conversion, as the LE state is less energetically favourable than the CT state.

To confirm the nature of these LE and CT states, we further conducted an electron–hole analysis of these dyes using the Multiwfn program, by obtaining and comparing the D_CT index (Table S1, ESI†).^40–42 The D_CT index measures the distance between the centres of mass of electrons and holes, whereby a larger D_CT index corresponds to a higher degree of CT. For DRHN1, upon geometrical relaxation, its D_CT index is 1.510 Å in the LE state, as the hole and electron are mainly located at the xanthene scaffold (Fig. 1b). In contrast, complete charge separation occurs between the xanthene scaffold and phthalimidine moiety in the CT state with the D_CT index rising to 4.545 Å.

Subsequently, we simulated other rhodamine dyes (in the closed form). Their corresponding energy levels, as well as electron and hole distributions, were calculated (Fig. S1 to S12, ESI†). The results are similar to that of DRHN1. A low-lying CT state (S₁) appears in all these compounds upon excited-state geometry relaxations, and these CT states all afford a small oscillator strength (<0.1, most of them approach zero, Fig. 1c). Consequently, the energy of the excited state of these rhodamines would be released from the CT state mainly via non-radiative decays.

We also calculated the energy gap between the LE state and the CT state (ΔE_LE–CT) (Fig. 1d). ΔE_LE–CT reflects the tendency of state-crossing between the “bright” LE state and the “dark” CT state. Should ΔE_LE–CT becomes negative, the LE state will become the most stable excited state, thus activating bright fluorescence from the closed-form rhodamines. Nevertheless, in all rhodamines under study (with varied heteroatom replacement, donor substitution, and spirocyclic ring locking groups), ΔE_LE–CT remains positive; the dark CT state remains the most stable excited state. These computational results are consistent with the extremely weak fluorescence of the closed-form rhodamines.

To verify the accuracy of our calculations, we compared the computational results of DRHN14 (or Rhodamine 101) with experimental data reported by Karpiuk.¹³ Experimental measurements showed that the dipole moment in the excited state of the rhodamine 101 was ∼26 D with a distance of full charge separation over 5 Å. Our calculated dipole moment and the D_CT index of DRHN14 are 26.671 D and 5.135 Å (Table S1, ESI†), respectively. This good match with the experimental data demonstrates the reliability of our computational results. Furthermore, we compared the calculated results of DRHN14 using M062X and ωB97XD with def2SVP (Fig. S13 and Table S2, ESI†). The same conclusion can be obtained by different functionals, with slight variations in calculated energy levels.

Henceforth, we will denote the CT state (with charge transfer from the aniline group to the meso-substituent) as CT₁. This is to differentiate with a newly arising CT state in the next section.

Expanding the π-conjugation of the xanthene moiety may not activate fluorescence in the closed-form rhodamine analogs

We next considered how to activate fluorescence in the closed form of “rhodamines”. The activation of fluorescence in the closed form of rhodamine analogs requires the stabilization of the “bright” LE state, and/or the destabilization of the “dark” CT₁ state. This is to ensure that the LE state becomes the most stable state in the adiabatic excited state (AES). To achieve this goal, we may expand the π-conjugation of the xanthene moiety to lower the energy level of the LE state. Inspired by this strategy, we modelled two dyes, namely BRHN2 and BRHNBO (Fig. 2).

Fig. 2 Molecular structures, energy levels in the adiabatic excited states, and the corresponding electron and hole distributions of (a) BRHN2 and (b) BRHNBO in water.

In BRHN2, the extended π-conjugation of the amino-anthracene fragment (in comparison to the amino-phenyl group in conventional rhodamines) leads to a more stable LE state (2.577 eV) with a larger oscillator strength (f = 0.260), compared with the LE state (4.245 eV) in DRHN1. The LE state of BRHN2 mainly involves the photoexcitation of the amino-anthracene fragment and is more stable than the CT₁ state (3.174 eV) in BRHN2. The negative ΔE_LE–CT of −0.597 eV in BRHN2 indicates that BRHN2 is emissive even in the closed form. It is worth highlighting that the stable LE state in the closed form of BRHN2 also indicates a visible peak emission wavelength.

In contrast, our results showed that BRHNBO remains dark, despite the expanded π-conjugation. In BRHNBO, the LE state (2.462 eV; f = 1.218) is much more stable than the CT₁ state (3.222 eV; f = 0.055), due to the incorporation of a large π-conjugated BODIPY fragment. However, a new CT state (CT₂: 1.533 eV; f = 0.002) appears, due to electron transfer from the anilino group to the newly installed BODIPY moiety. CT₂ is the most stable excited state and is much more stable than the LE state with an energy gap of ΔE_LE–CT = 0.928 eV in AES. Because of this low-lying CT₂, BRHNBO remains dark in the lactam structure.

We also modeled the CT₂ state in BRHN2. Although the energy of this CT₂ state is lower than that of the CT₁ state, the CT₂ state is less stable than the LE state. BRHN2 thus remains emissive even in the closed form.

These computational results collectively show that expanding π-conjugation plays an important role in stabilizing the LE state with respect to CT₁ but does not necessarily turn on fluorescence. To do so, we also need to suppress the formation of the low-lying CT₂.

Employing frontier molecular orbitals (FMOs) to screen dual-emission rhodamine analogs

To rapidly design rhodamine analogs with the low-lying LE and high-lying CT₁ and CT₂, we turned our attention to the frontier molecular orbitals (FMOs) of various moieties in these compounds. For example, due to the disrupted π-conjugation, DRHN1 can be divided into three moieties, including M1, M2, and M3 (Fig. 3a). The energy levels of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in hydrogen passivated M1, M2 and M3 were subsequently calculated. Based on the energy levels of these FMOs, one can quickly identify the prompt occurrence of CT₁ from M2 (or M1) to M3, upon the LE photoexcitation of M2 (or M1). This is because the LUMO of M3 has a lower energy level than that of M2 (or M1) with a difference of 1.07 eV. This configuration of the FMO energy levels satisfies the requirement of donor-type photoinduced electron transfer (d-PET) from M2 (or M1) to M3.⁴³ This efficient d-PET process (corresponding to the low-lying CT₁ state) could effectively quench the fluorescence of DRHN1.

Fig. 3 M1, M2, and M3 moieties and their corresponding HOMO and LUMO energy levels in the ground state in (a) DRHN1, (b) BRHNBO, and (c) BRHN2 (L: LUMO; H: HOMO).

A similar analysis was performed on the FMOs of three moieties in BRHNBO (Fig. 3b). In this case, upon the LE photoexcitation of M2, acceptor-type PET (a-PET) could readily occur from M1 to M2, as the HOMO of M1 has a higher energy level (−6.651 eV) than that of M2 (−7.200 eV). The efficient electron transfer from M1 to M2 corresponds to the formation of the stable CT₂ state. The stable CT₂ state could quench the fluorescence of the closed form of BRHNBO, rendering it non-emissive.

Finally, we investigated the FMOs in the three fragments of BRHN2 (Fig. 3c). The energy levels of the FMOs in M2 reside between those of M1 and M3. Such an alignment effectively inhibits charge transfer between M2 and M1/M3. In other words, the LE state of M2 in BRHN2 remains the most stable excited state (S₁). The closed form of BRHN2 is thus highly emissive.

Predictions based on FMOs are consistent with our previous calculations based on the “optical” gaps (the relative energy levels between various excited states). Compared to the extensive excited-state calculations, FMOs can be rapidly obtained via simple ground state calculations on small molecular fragments. The approximation using FMOs thus offers a highly convenient method to design dual-emission rhodamine analogs.

Validation of the FMO-based screening method in developing dual-emission rhodamine analogs

Inspired by the success of the FMO-based screening method, we next optimized and calculated the HOMO and LUMO energy levels of 48 moieties (Fig. 4). Panel A includes eight potential fragments to form M3; Panel B includes forty candidates of M1 and M2. By matching the energy levels of FMOs, a judicious choice of M1, M2, and M3 moieties could collectively form dual-emission rhodamine analogs. To this end, we also modelled the FMOs of these fragments using B3LYP and M06-2X with the def2SVP basis set in water (Fig. S14, ESI†). The relative energy differences between the calculated FMOs are consistent with those derived from CAM-B3LYP.

Fig. 4 (a) Candidates of M1, M2, and M3 fragments in rhodamine analogs. (b) Calculated electronic gap in the ground state of these fragments in (a) using CAM-B3LYP/def2SVP in water.

To demonstrate the application of this FMO-based screening method, we selected F1, F27, and F40 to serve as M1, M2, and M3, respectively (Fig. 5a). The energy levels of the FMOs in F27 are sandwiched between those of F1 and F40. This energy alignment inhibits both d-PET and a-PET between M2 and M1/M3, ensuring bright emissions from the LE state of M2. This prediction is further validated by the extensive calculations of the AES in the resulting derivative BRHN5 (Fig. 5b). In BRHN5, the LE state is much more stable than CT₁ and CT₂ by 0.542 eV and 0.593 eV, respectively, indicating strong fluorescence in the visible region from the closed form. As expected, the open form of BRHN5 is also highly emissive (f = 1.352), making this compound a dual-emission compound (Fig. S15, ESI†).

Fig. 5 (a) Three fragments, their respective HOMO and LUMO energy levels in the ground state, and (b) the corresponding distributions of electrons and holes, and the energy levels of adiabatic excited states of BRHN5. (c) Three fragments, their respective HOMO and LUMO energy levels in the ground state, and (d) the corresponding distributions of electrons and holes, and the energy levels of adiabatic excited states of BRHN4. Molecular structures of (e) dual-emission rhodamine analogs and (f) single-emission rhodamine analogs (in the open form only).

We also constructed a control compound BRHN4 (Fig. 5c). BRHN4 is formed by joining three fragments, F1 (M1), F26 (M2), and F40 (M3). The HOMO energy level of F1 is higher than that of F26 by 0.34 eV, indicating substantial d-PET from F1 to F26, thus quenching the emissions of BRHN4. As expected, detailed excited-state calculations showed that the dark CT₂ state is more stable than LE by 0.867 eV in BRHN4, rendering it non-emissive (Fig. 5d).

By matching the energy levels of HOMOs and/or LUMOs, we further designed several bright and dark closed-form rhodamine analogs (Fig. 5e and f, respectively). Subsequent excited state calculations and the distributions and energy levels of the molecular orbitals verified the accuracy of the FMO-based design method (Fig. S16–S38, ESI†). Their D_CT indices are presented in Table S1 (ESI†). Moreover, the bright rhodamine analogs in the open form are all highly emissive with large oscillator strength as suggested by the calculation results (Fig. S39–S43, ESI†). It is of note that an analog of ProbeAC has been reported to exhibit dual emissions, corroborating the accuracy of our computational designs.⁴⁴

Guided by this FMO-based screening method, chemists could facilely design and screen rhodamine analogs with closed-form emissions, instead of trials-and-errors. Given the open form of these derivatives are also highly emissive, the spirocyclization reactions would enable dual emissions. Nevertheless, since this method ignores exciton binding and solvent effects in stabilizing various excited states, environment-dependent variations in fluorescence intensities in the closed form of these rhodamine analogs are expected in some cases.

Potential applications of dual-emission rhodamine analogs

Dual-emission materials hold great potential for various applications.^45,46 In rhodamine analogs, the additional emission band in their closed form could enable many new applications, such as building ratiometric fluorescent probes and fluorophores for super-resolution imaging (Scheme 1b). Currently, a common strategy to build ratiometric probes based on rhodamines requires the employment of one additional fluorophore (i.e., via the Förster resonance energy transfer mechanism).^47,48 Nevertheless, the reliability of such ratiometric probes is compromised, as these dyes have different photostability and their relative concentrations may vary over time (due to photobleaching). This problem can be addressed via the dynamic spirocyclic equilibrium of dual-emission rhodamine analogs.

Dual-emission rhodamine analogs also hold great promise in performing simultaneous conventional and super-resolution bioimaging.^49,50 Conventional fluorescence imaging offers fast imaging acquisition speed but low optical resolution. In contrast, super-resolution imaging (i.e., based on single-molecule localization) breaks the diffraction limit and provides ∼20 nm lateral resolution, but at the expense of long imaging acquisition time. Additionally, single-molecule localization techniques require “sparse emissions” and “blinking” in organic fluorophores to construct high-resolution images; such fluorescent properties often demand tedious optimization of imaging buffers or additives. In existing rhodamines with one emission band, conventional imaging and super-resolution imaging cannot be conducted at the same time. However, the dual-emission rhodamine analogs could make the simultaneous conventional and super-resolution imaging possible: the short-wavelength emissions from the closed form (the majority) could be employed in conventional imaging to provide a global view of the sample under study, while the sparse long-wavelength emissions (the minority) enable super-resolution imaging in regions of interests. The blinking of these rhodamine analogs could either occur spontaneously or be controlled via photoactivation.⁵¹ Due to the expanded π-conjugation and the red-shifted UV-vis absorption in the closed form, these new rhodamine analogs could also greatly reduce phototoxicity in comparison to conventional photoactivatable rhodamines.

Conclusions

In conclusion, using computational modelling and calculations, we uncovered the mechanistic underpinnings that explain why the closed form of rhodamines is weakly emissive: the energy level of the (nearly) non-emissive CT state is lower than that of the bright LE state upon excited-state geometry relaxation. Utilizing this knowledge, we proposed a facile yet effective design strategy to develop dual-emission rhodamine analogs by selecting fragments with matched FMO energy levels to avoid inter-fragment photoinduced electron transfer. This strategy could stabilize/destabilize the LE/CT states, thus activating dual-emission bands from both the closed and open forms of rhodamine analogs. These dual-emission fluorophores endow exciting new opportunities for advancing bioimaging and biosensing applications.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work is supported by the Agency for Science, Technology and Research (A*STAR) under its Advanced Manufacturing and Engineering Program (A2083c0051), the Ministry of Education, Singapore (MOE-MOET2EP10120-0007), the National Natural Science Foundation of China (22225806, 22078314, 21878286, 51902124), the Research Start-up Fund Project of Hainan University (No. RZ2200001217) and the Tianjin University-Hainan University Independent Innovation Fund (RZ2200003795). The authors are grateful for the computing service of SUTD-MIT IDC and the National Supercomputing Centre (Singapore).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2qm01351g

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